En Dual Tracer PET-MR-protokollet för den kvantitativt mått på regional Brain energisubstrat Upptag i Rat

Summary

Small-animal positron emission tomography enables the assessment of the brain's two main energy substrates: glucose and ketones. In the present method, ¹¹C-acetoacetate and ¹⁸F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

Abstract

We present a method for comparing the uptake of the brain's two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which ¹¹C-AcAc and ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of ¹¹C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of ¹¹C-AcAc and ¹⁸F-FDG brain uptake (cerebral metabolic rate; μmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases.

Introduction

Context and Rationale

Positron emission tomography (PET) enables the minimally-invasive study of functional processes in the brain. Glucose is the brain's main energy substrate, but in conditions of glucose deficiency, ketones (acetoacetate [AcAc] and β-hydroxybutyrate) are the main alternative energy substrates. Brain energy metabolism has been widely studied by PET using the most common PET tracer, ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG), a glucose analog. Our group recently developed a novel radiotracer –¹¹C-AcAc – to measure brain ketone metabolism¹. Magnetic resonance imaging (MRI) is a much higher resolution technique (0.1 mm × 0.1 mm in-plane resolution) than PET, and is needed to clearly localize anatomical brain regions required for the regional PET analysis of brain energy metabolism.

PET data are commonly expressed as standardized uptake values (SUV)^2-6. SUV are the tissue activity concentration normalized by the fraction of the injected dose/unit weight, as initially proposed over 70 years ago⁷. These units are still widely used because they require simpler PET acquisition and image analysis methodologies. However, an important limitation is that SUV are relative not absolute units, making it difficult to compare results across different studies. This difficulty of comparison may contribute to contradictory findings in the literature on brain glucose uptake in the elderly⁸.  Therefore, the quantitative cerebral metabolic rate (CMR; μmol/100 g/min) has particular advantages^9-11. Generating CMR values requires a dynamic PET acquisition and the plasma radioactivity counts as a function of time, i.e. the plasma time-activity curve (TAC) or input function. The input function can be obtained by multiple blood samplings throughout the PET acquisition^9,12 or by the image-derived input function (IDIF) method, in which a region of interest is drawn on a blood pool (heart's left ventricle or major artery)^11,13-16.

Goal

The aim of our method was to quantitatively compare for the first time the uptake of the brain's two key energy substrates, glucose and ketones, using PET and MRI in rodents. ¹¹C-AcAc and ¹⁸F-FDG were used sequentially in the same animal. The protocol was designed to measure regional uptakes in different relevant brain structures (cortex, hippocampus, striatum, and cerebellum) clearly visible in the MR images. The protocol was also specifically intended to permit quantitative analysis of tracer brain uptake, i.e. CMR of both ¹⁸F-FDG and ¹¹C-AcAc. Although this protocol was developed to study brain energy substrates, the radiotracers we used could be replaced by others, and the same methodology can be used to study different brain functional processes.

Advantages over existing methods

PET and MRI do not require the animal to be sacrificed after the acquisition. Therefore, follow-up studies of treatments are possible. Thus, baseline data followed by an experimental condition can be measured within the same animal, thereby reducing both biological variability and the number of animals required. A key advantage of our dual tracer PET protocol is to compare both tracers uptake in the same animal under the same physiological conditions within the same imaging session, thereby reducing even more biological variability and systematic discrepancies. This dual tracer PET protocol is feasible primarily because of the short physical half-life of ¹¹C (20.4 min) and the fast biological washout of ¹¹C-AcAc, which leave minimal residual ¹¹C radioactivity during the second acquisition with ¹⁸F-FDG. The MRI scan is an important feature of this protocol as it enables the tracer uptake to be studied in specific brain areas. In addition, this method enables an absolute quantitative measure of brain tracer uptake in contrast to relative units obtained by the SUV method. Finally, ¹¹C-AcAc images have a low signal-to-noise ratio because of relatively low brain AcAc uptake under physiological conditions, which makes automatic ¹¹C-AcAc and MR images registration challenging. Hence, because ¹¹C-AcAc and ¹⁸F-FDG acquisitions are sequential (no motion of the animal), the ¹⁸F-FDG to MRI alignment can be applied to ¹¹C-AcAc images.

Key papers where the protocol has been used

We have used the dual tracer PET protocol in a study involving the comparison of the fasted state and the ketogenic diet (KD) in young rats². We showed that both fasting and the KD increase significantly both ¹¹C-AcAc and ¹⁸F-FDG brain uptake. However, these were not quantitative results as we did not use the dynamic PET imaging and tracer kinetic modeling methodology at that time. Thereafter, we undertook a regional and quantitative study of brain metabolism in aged rats, where the effect of aging and a KD were evaluated on brain ¹¹C-AcAc and ¹⁸F-FDG uptake¹¹. We also showed that the percentage of distribution across brain regions was different between ¹¹C-AcAc and ¹⁸F-FDG. Furthermore, not only the CMR of ¹¹C-AcAc but also that of ¹⁸F-FDG was increased in the whole brain as well as in the striatum of aged rats on the KD.

Protocol

All experiments were completed in accordance with the Animal Care and Use Committee at the Université de Sherbrooke and with the Canadian Council on Animal Care. The experimental protocol was approved by the Institutional Animal Research Ethics Review Board (protocol #011-09). 1. Brain Anatomy with MRI Let rats acclimatize in the animal facility for a minimum of 7 days prior to the protocol. Perform brain MRI scans 1-2 weeks prior to the dual tracer PET protocol to allow …

Representative Results

As seen in Figure 2,11C-AcAc uptake is low within the brain itself. As mentioned earlier, ketones consumption by the brain is very low on a short-term fasting. 11C-AcAc uptake is higher in the tongue and cheek muscles. Indeed, ketones are rapidly taken up by rat skeletal muscles23. In contrast, 18F-FDG uptake is mostly in the brain and the cheek muscles. Figure 2 shows that during the coregistration process, MR images are fixed and PET ima…

Discussion

Critical steps

A critical step in this dual tracer PET protocol is to be able to simultaneously scan the heart's left ventricle and the brain at the same time. This requires a PET scanner with a sufficient axial length, i.e. a minimum of 7.5 cm. A few test scans are needed to determine the exact position of the scanner table (x, y, and z values), where the brain and the heart are scanned correctly.

Tracer injection is also a crucial point for a succes…

Materials

MRI scanner	Varian		7 Tesla
Small-animal PET scanner	Gamma Medica		Lab-PET/-Triumph
Heat mat	Sunbeam	PN 143937
Heater system	SA Instruments	761 100 Rev B
Respiratory gating	SA Instruments	SAII's P-resp
Clinical chemistry analyzer	Siemens Healthcare Diagnosis	765000.931	Dimension Xpand Plus
Polyethylene tubing 50	Becton Dickinson	427411
Injection pump	KD Scientific	Model 210
Gamma-counter	GMI	Packard Cobra II
Centrifuge	Thermo Scientific	75002416	Heraeus Pico 21
PMOD software	PMOD Technologies		PMOD 3.2 version
Geiger counter	Fluke Biomedical	ASM-990	Advanced Survey Meter
Reagent
Name of the Reagent	Company	Catalogue Number	Comments (optional)
Isoflurane	Abbott Laboratories, Ltd	B506
0.9% NaCl solution	Hospira	4888010
Heparin	Sandoz	1004336
Isopropenyl acetate	Aldrich	11778	99%
Methyllithium	Aldrich	197343	1.6 M
THF	Aldrich	87371
Flex reagent cartridge glucose	Siemens Healthcare Diagnosis	DF40
Trizma base	Sigma	T6066-500G	prepare tris buffer 100 mM pH 7.0
Sodium oxamate	Sigma	O2751	20 mM
NADH	Roche	10128015001	0.15 mM
b-Hydroxybutyrate dehydrogenase	Toyobo	HBD-301	1 U/ml